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Original Studies |
Anatomisches Institut der Technischen Universität München (A.M., S.B.), D-80802 München, Germany; Departments of Anesthesiology and Pharmacology (H.C.H.), Cornell University Medical College, New York, New York 10021; The Rockefeller University (G.L.S., P.G.), New York, New York 10021; and I. Frauenklinik der Ludwig-Maximilians Universität München (U.B., C.B.), D-80337 München, Germany
Address all correspondence and requests for reprints to: Artur Mayerhofer, M.D., Professor of Molecular Anatomy, Anatomisches Institut, Technische Universität München, Biedersteiner Strasse 29, D-80802 München, Germany. E-mail: mayerhofer{at}lrz.tum.de
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Abstract |
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- and ß-adrenergic receptors and modulates
ovarian steroidogenesis, but the significance of ovarian DA is unclear.
We examined whether a DA receptor of the D1-subtype (D1-R) is present
in human ovary and in cultured human granulosa luteal cells (GC). Using
RT-PCR, we cloned complementary DNAs from adult human ovarian and GC
messenger RNAs, which are identical to the human D1-R sequence. In
ovarian sections, D1-R protein was identified (by immunohistochemistry)
in granulosa cells of large antral follicles, cells of the corpus
luteum, as well as in cultured GC. An immunoreactive band of
approximately Mr 50,000 was found in cultured luteinized GC using the
same antiserum in Western blots. The D1-R in these cells was
functional, because DA, alone or in the presence of the ß-receptor
antagonist propranolol, caused cellular contraction. The selective D1-R
agonist SKF-38393 induced a similar change in cytomorphology and
increased the levels of media cAMP. SKF-38393 failed, however, to
significantly affect basal and hCG-stimulated progesterone
release in vitro, indicating that the activation of the
D1-R was not directly linked to synthesis of progesterone,
the major steroid of human GC. Estradiol synthesis likewise was not
affected. Using RT-PCR and immunohistochemistry, we found that GC
express DA- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32),
a protein typically associated with neurons bearing the D1-R. In
cultured GC, DA and SKF-38393 induced increased
threonine-phosphorylation of DARPP-32, even in the presence of
propranolol but not in the presence of D1-R antagonist SCH-23390. Taken
together, the presence of DA and a functional DA receptor and DARPP-32
indicate that a novel, physiological regulatory pathway involving DA
exists in the human ovary. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-adrenergic receptors exist on
steroidogenic cells in many species, including human (8), and
activation of these receptors by NE can affect steroid production.
Interfering with the normal development of ovarian innervation by
neonatal denervation led to abnormal ovarian development and function
(9), and induction of polycystic ovaries in rats was associated with
hyperactivation of the ovarian catecholaminergic system (10). More
recently, we showed that neurotransmitters, acting via ß-adrenergic
ovarian receptors and cAMP, are responsible for the initiation
of FSH-receptor expression and follicular growth in the neonatal rat
ovary (11). Ovarian catecholamines in mammalian species reach the ovary from the adrenal via the blood stream or can be released from the sympathetic innervation of the ovary (12). Catecholamines may be of greater importance for the regulation of gonadal function in primate species, such as the rhesus monkey, which possess an additional intraovarian and intratesticular potential source of catecholamines, namely neuron-like cells (13, 14). These cells are immunoreactive for neuronal markers, including neurofilaments and the low affinity receptor for nerve growth factor, and for tyrosine hydroxylase, the rate-limiting enzyme of the catecholamine biosynthetic pathway. Moreover, gonadal tyrosine hydroxylase gene expression was demonstrated in the rhesus monkey (15). These neuron-like cells, in conjunction with the extrinsic innervation and catecholamines derived from the adrenal, could be the sources of ovarian NE and dopamine (DA).
DA is documented in the ovary (16, 17), and high levels are reported, in particular, in antral fluid of preovulatory human follicles (18); but the physiological function of DA in the ovary is not clear. It has been assumed that DA may serve as a precursor for the biosynthesis of NE (19). In a previous report, which describes effects of DA and NE on steroid production by cultured human granulosa cells (GC) (20), such a precursor role was suggested. The effects of DA on steroid production were inhibited by the ß-receptor antagonist propranolol and, therefore, did not indicate a direct action of DA, but suggested action of NE on ovarian ß-adrenergic receptors. Direct actions of DA within the ovary would require specific DA receptors, a possibility not examined in human or primate ovary. However, in cell cultures derived from pregnant mare’s serum gonadotropin-treated immature rat ovaries, specific binding of DA agonists was found, indicating the presence of a DA receptor (21). In addition, a stimulatory effect of DA and, in particular, of DA-1 receptor (D1-R) agonists, on progesterone production was demonstrated, further supporting the existence of a functional receptor of the D1-subtype in this system. Though providing indirect evidence for the existence of ovarian D1-R, these studies do not allow conclusions as to the physiological relevance or cellular site(s) of DA receptor expression in vivo.
In the present study, we used RT-PCR, Western blotting, and immunocytochemical techniques to determine whether DA receptors of the D1-R type are present in the human ovary. Using cultured human granulosa luteal GC cells, we also studied the functional relevance of these receptors and examined if DA- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) is expressed and regulated in these cells.
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Materials and Methods |
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Follicular fluid, containing GC, was derived from in vitro fertilization (IVF) patients stimulated according to routine protocols. The experimental procedure and the use of the cells were approved by the local ethics committee, and the women gave written consent. Isolation and culture of the cells were performed as described previously (8, 22, 23, 24, 25), with slight modifications. These include a Percoll centrifugation step (50%, Pharmacia, Freiburg, Germany; according to Ref. 26), which was performed to avoid red blood cell contamination. Cells were washed with DMEM:Ham’s F12 (1:1) and seeded in the same medium with 10% FCS onto 35- or 60-mm Falcon (Nunc, Wiesbaden, Germany) culture dishes (coated with laminin (Sigma Chemical Co., Deissenhofen, Germany), as described (8, 22, 23, 24, 25), and in some cases, also on Labtek cell culture chambers (Nunc). Cultures were incubated in a humidified atmosphere with 5% CO2 at 37 C. After 24 h, media were replaced, and nonadherent cells (mainly red blood cells) were removed by gentle washing. Cells were used at this time, for most experiments or, in some cases, were kept longer, with medium changes every other day.
Paraffin-embedded normal ovarian tissue samples were obtained from a tissue archive at the Women’s Hospital in Munich. These samples had been collected from eight premenopausal women during autopsies. All samples had been fixed in formalin and were embedded in paraffin for initial pathological evaluation. For the retrospective evaluation, sections (5 µm) from these blocks were cut and used for immunohistochemistry, as described below.
RT-PCR cloning
Total RNA from several batches of cultured GC (1 day after isolation) was prepared using an RNEasy kit (Qiagen, Hilden, Germany). We used 200–500 ng total RNA for RT, using an 18-mer polydeoxythymidine primer and Moloney’s murine leukemia virus reverse transcriptase (11, 27). In addition, a commercial human complementary DNA (cDNA; 2 µL), reverse transcribed from pooled adult ovarian messenger RNA (mRNA), was used for PCR (Invitrogen, DeSchelp, The Netherlands). The primers for the PCR amplification of D1-R were both 19-mers, complementary to regions of the coding sequence of the human D1-R mRNA (28), namely nucleotides (nt) 1084–1102 (sense primer: 5'-CTG AAG ACT CTG TCG GTG A-3') and nt 1341–1359 (antisense primer: 5'-ACT CAC CGT CTC TAT GGC A-3'). For DARPP-32 PCR, we used two oligonucleotide primer sets homologous to regions of the bovine DARPP-32 sequence (29), with some modifications, as marked by underlining (Ehrlich et al., unpublished sequence information), namely a sense primer (21-mer: 5'-CTG TGC CTA CAC ACC ACC TTC-3') corresponding to nt 554–574, and an antisense primer (25-mer: 5'-GCC AGT CCA TCT TGC AGG CAC CCA G-3') corresponding to nt 1491–1515. For a nested, second PCR amplification, another pair of 18-mer oligonucleotide primers homologous to the bovine DARPP-32 sequence were used, namely sense primer: 5'-ATT GCT GAG TCT CAC CTG-3' (nt 594–611), and antisense primer: 5'-TCC ACT TGG TCC TCA GAG-3' (866–883).
PCR reactions were carried out as described (11, 27). PCR amplifications consisted of 35 cycles of denaturing (94 C, 15 sec), annealing (55 C, 1 min), and extension (72 C, 2 min). The PCR reaction products were separated on 2% agarose gels and visualized with ethidium bromide. D1-R cDNAs and DARPP-32 cDNAs were subcloned into the pGEMT vector (Promega Corp., Mannheim, Germany) and were sequenced using a fluorescence-based dideoxy sequencing reaction. An automated sequence analysis was performed on an ABI model 373A DNA sequencer (Perkin-Elmer, Überhingen, Germany).
Immunohistochemistry
The cellular distribution of D1-R protein in the ovaries and in cultured GC was determined by immunohistochemistry using a commercially available polyclonal antiserum (rabbit anti-D1-R, R&D Systems, Wiesbaden, Germany, 1:500–1:1,000). Immunocytochemistry, using a well-characterized monoclonal mouse anti-DARPP-32 antibody (30, 1:2,000–1:5,000), was also performed in cultured GC. Immunocytochemical and immunohistochemical procedures, using the avidin-biotin (ABC)-method and immunofluorescence methods, were employed as described (14, 31). In the case of the GC, cells were fixed on slides (Zamboni’s fixative) and used directly after rinses in 0.01 mol/L PBS (pH 7.4). Sections were first deparaffinized, and endogenous peroxidase reactivity was quenched by a 10-min pretreatment with 10% methanol, 0.3% H2O2 in PBS, followed by a 5-min incubation with 0.5% Triton X-100 in PBS and subsequent incubation with normal goat serum (5% in PBS, 30 min). Incubation with the antiserum/antibody was carried out overnight in a humidified chamber. A biotinylated secondary antiserum (goat antirabbit IgG, or goat antimouse IgG; Camon, Wiesbaden, Germany, 1:500 diluted in PBS with 1% BSA) and a commercial ABC kit (Vectastain; Camon) were used. Immunoreactivity was visualized with 0.01% H2O2 and 0.05% diaminobenzide solution (in 0.05 mol/L Tris-HCl, pH 7.6). In some cases, fluorescein isothiocyanate-labeled secondary goat antirabbit antiserum was used instead of the ABC-detection (14, 31). For control purposes, the first antiserum/antibody was omitted, and incubations with normal rabbit serum/mouse serum were carried out instead. Sections were examined with a Zeiss Axiovert microscope (Zeiss, Oberkochen, Germany), equipped with a fluorescein isothiocyanate filter set.
Western blotting
Western blotting was performed as previously described (24, 31), with minor modifications. In brief, cells were harvested, frozen, thawed, homogenized in 62.5 mmol/L Tris-HCl buffer (pH 6.8) containing 10% sucrose and 2% SDS, sonicated, and heated (95 C for 5 min) in the presence of 10% mercaptoethanol. Samples (15 µg/lane) were separated electrophoretically on 10% or 12.5% SDS-polyacrylamide gels (SDS-PAGE). Proteins were transferred onto nitrocellulose membranes and probed with the same D1-R antiserum used for immunohistochemistry (1:1,000 dilution, incubation overnight at 4 C).
In addition, a well-characterized monoclonal phospho-DARPP-32 specific antibody was used (1:500) (32) to examine whether treatment of GC (for 1 h, in 2 cases, also 24 h) with DA (Sigma Chemical Co., 1 and 10 µmol/L) or SKF-38393 (1 and 10 µmol/L, RBI, Biotrend, Cologne, Germany, diluted in medium without serum) changed the phosphorylation of DARPP-32. For control purposes, the ß-receptor antagonist propranolol (Sigma Chemical Co., 10 µmol/L) or the D1-R antagonist SCH-23390 (10 µmol/L, RBI) were added to the cells treated with DA or SKF-38393 (used at 1 µm). Cell morphology was monitored and documented. Immunoreactivity was detected using peroxidase-labeled antisera (1:3,000, Dianova, Hamburg, Germany) and enhanced chemiluminescence, as described (33) (Amersham Buchler, Braunschweig, Germany). In some cases, the blots were digitized, and integrated optical densities of the bands were determined using an edited version of the program NIH Image, as described previously (11).
Immunoprecipitation experiments
Immunoprecipitation experiments were carried out to examine whether SKF-38393 (100 µmol/L, diluted in medium without serum) treatment of cultured GC (1 day after isolation) for 1 h increased threonine phosphorylation of DARPP-32. Media were removed, and cells were solubilized in buffer, containing 10 mmol/L NaH2PO4, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% Triton X-100, 0.25% SDS, 1% sodium deoxycholate, and 2 mmol/L phenylmethanesulfonylfluoride. For immunoprecipitation, we used magnetic beads labeled with antimouse IgG (Dynabeads, Dynal, Hamburg, Germany) and magnetic separation. The beads were first incubated with normal mouse serum (5% in PBS containing 10 mmol/L EGTA, 250 mmol/L saccharose, and 0.1% BSA) and were then labeled with 2 µL of the well-characterized monoclonal antibody directed against bovine DARPP-32, which recognizes primate DARPP-32 and which was used for immunocytochemistry, as well (30). Subsequently, beads were incubated with 150 µL GC cell lysate for 1 h at room temperature and then for 30 min at 4 C. After magnet separation, pellets were washed several times and used for SDS-PAGE, as described. Blots were developed using a monoclonal antibody against phospho-threonine (Sigma Chemical Co., 1:100); and, in some cases, they were evaluated densitometrically (see above) (11).
Progesterone and estradiol measurements
The release of progesterone and estradiol into the culture medium by GC incubated for 6 h with SKF-38393 (10 µmol/L) in the absence or presence of hCG (10 IU/mL, Sigma Chemical Co.) was examined using triplicate wells for each treatment (n = 3). Samples were analyzed using commercial enzyme immunoassays (Biochem Immunosystems, Freiburg, Germany), following the instructions of the manufacturer. Intraassay coefficients of variation ranged between 5–8%, and interassay coefficients of variation did not exceed 10%. All incubation and pipetting steps and the calculations of hormone concentrations were carried out in a fully automated immunodiagnos-tic analyzer (Labotech Automatic Immunodiagnostic Analyzer, Chemila, Rome, Italy). Results were corrected for small changes in cellular protein (BCA method, Pierce, Rockwell, IL). ANOVA and t test were used to evaluate the results.
Determination of cAMP
The levels of cAMP in the media of GC, 1 day after isolation, were examined after incubation for 3 or 6 h with SKF-38393 (1–100 µmol/L) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (1 mmol/L, Sigma Chemical Co.). In a pilot study, SKF-38393 (at a concentration of 1 µmol/L) caused a small, but not statistically significant, increase in cAMP (20% over basal levels). Therefore, for three independent additional experiments, a higher SKF-38393 concentration (100 µmol/L) was used. These samples were measured using an enzyme immunoassay (R&D Systems), according to the instructions of the manufacturer. The sensitivity of the assay was 0.5 pmol/mL, and the intraassay coefficient of variability was smaller than 10%. To correct for small differences in cell density, cAMP results were expressed per microgram of cellular protein (BCA method, Pierce). Student’s t test was used to evaluate data.
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Results |
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RT-PCR amplification yielded cDNAs of expected sizes (276 bp),
which (upon subcloning and sequencing) were found to be identical to
the human D1-R-gene sequence (three identical clones from the human
ovary and three identical clones from human IVF patient-derived
luteinized GC were analyzed; Fig. 1
).
Ovarian D1-R protein was demonstrated in paraffin-embedded human
ovarian tissue using immunohistochemistry and in GC using
immunocytochemistry. D1-R protein was found in granulosa cells of large
follicles (approximate antral diameter, 3–10 mm) and in luteal cells
of the corpus luteum (Fig. 2). Staining
seemed homogeneous within these cells. In GC, D1-R immunoreactivity was
associated with the cell membrane and/or with the cytoplasm of most
cells, but some cells seemed not to be immunoreactive (Fig. 3). All controls performed were negative.
Using the same antiserum in Western blots, an immunoreactive band of
approximately Mr 50,000, i.e. in the range of the expected
size of the D1-R (Mr 49,000), was found in IVF-derived GC early after
isolation, which persisted during several days in culture (Fig. 4).
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The D1-R in GC cultures was biologically active, because SKF-38393
(100 µmol/L) significantly increased cAMP levels (2- to 3-fold) in
cultured human luteinized GC within 3–6 h (Fig. 5). DA, at 1 µmol/L, induced a marked
contraction of GC (Fig. 6), which was not
affected by adding propranolol (10 µmol/L, not shown) but was
mimicked by SKF-38393 (1 µmol/L, not shown). Activation of the
ovarian D1-R in our system was not directly linked to the synthesis of
progesterone or estradiol in human luteinized GC within
6 h. The selective D1-R agonist SKF-38393 (10 µmol/L) failed to
reliably affect basal and hCG-stimulated progesterone
release in vitro in three independent experiments (not
shown). A tendency to a decrease of progesterone after the
addition of SKF-38393 was apparent but was not statistically
significant. Results of estradiol measurements, likewise, showed no
statistically significant changes (data not shown).
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DARPP-32 was expressed in GC (three independent clones) and in
human ovary (one clone) as shown by RT-PCR amplification, subcloning,
and sequencing (Fig. 7). All four cDNA
clones (295 bp) were identical and showed 87% homology between the
human ovarian DARPP-32 and bovine DARPP-32 sequences (29) at the nt
level. The deduced amino acid sequence differs in two positions from
the one previously described (33): at position 155, arginine is changed
to cysteine; and at 157, leucine is changed to glutamine. DARPP-32, at
the protein level, was detected immunochemically in GC using a
monoclonal antibody (Fig. 8).
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) with a
specific-phospho-DARPP-32 antibody (32)], some variability was
apparent, depending on the batch of GC. In most experiments,
phosphorylated DARPP-32 was already observed under control conditions,
in the absence of DA or SKF-38393. This could be reduced by adding
SCH-23390 to 71% of control levels, as shown after densitometric
evaluation (two experiments). When GC cultures were treated with DA or
SKF-38393 (1 or 10 µmol/L) for 1 h, the
threonine-phosphorylation increased 1.3- to 5.15-fold over the basal
levels in four experiments. Thus, the mean increase was 248 ±
104% (means ± SEM). However, no change over the
already prominent basal levels in controls in two experiments was found
(Fig. 9; A, B, and C). D1-R antagonist SCH-23390 (10 µmol/L)
inhibited the SKF-38393 (1 µmol/L) effect on phosphorylation (SKF:
182% over control; SKF and SCH: 90% of basal level), whereas
coincubation with propranolol (10 µmol/L) did not (160% of control
levels; n = 2 for each group). An increased threonine
phosphorylation, induced by SKF-38393, was also observed after 24
h (two independent positive experiments, not shown). In most of these
blots, a doublet band became apparent, which most likely represents
additional phosphorylation of DARPP-32 at threonine 34 and at other
sites, presumably including serine 137 (32, 34, 35).
Threonine-specific phosphorylation of DARPP-32 by SKF-38393 (10
µmol/L, 1 h) was also demonstrated using a different approach,
namely, immunoprecipitation (using a monoclonal anti DARPP-32
antibody), followed by immunoblotting (using a phospho-threonine
antibody; four positive experiments; Fig. 9D). In these cases,
only a single band was observed. Densitometric analysis showed a 1.3-
to 6-fold increase [mean: 287 ± 120% (means ±
SEM)] in these experiments; these results are in the same
range as those detected with the other method used.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral D1-R gene expression has been found previously using
in situ hybridization techniques in another nonneuronal,
endocrine tissue, i.e. rat adrenal zona glomerulosa cells
(D1A-R) (37). These cells, like ovarian endocrine cells investigated in
the present study, are typical steroid-producing cells. In rat adrenal,
it was suggested (37) that DA might activate these receptors and
subsequently regulate adrenal steroidogenesis. Moreover, there is
evidence for an ovarian D1-R in cells derived from pregnant mare’s
serum gonadotropin-treated immature rat ovaries, based on specific
binding of a radioactive DA-antagonist (SCH-23390) (21). The authors
also demonstrated a stimulatory effect of D1-R agonists (including
SKF-38393) on progesterone and cAMP production by these
cells. The progesterone-stimulating effect was not
apparent in our study with GC, nor in another study with GC (20).
However, Bodis et al. (20) reported a modulatory, presumably
DA-induced effect on estradiol synthesis, a result abolished by the
ß-receptor antagonist propranolol. Thus, it is likely that this
effect is not a consequence of direct action of DA but rather is caused
by NE, to which DA may have been converted. NE subsequently may act on
ß-adrenergic receptors (8). Interestingly, pharmacological evidence
for a conversion of DA into NE has also been found in the bovine corpus
luteum (19). This fact and the known presence of - and
ß-adrenergic catecholamine receptors linked to modulation of
steroidogenesis in the ovary (8) may complicate interpretation of pure
pharmacological/functional studies dealing with effects of natural
catecholamines on steroid production.
In the present study, we minimized these complications by providing direct evidence for ovarian/GC D1-R gene expression using RT-PCR, Western blotting, and immunohistochemistry/immunocytochemistry. RT-PCR cloning showed the expression of the human D1-R gene in ovary and in GC. Its translated product, a protein of an expected Mr of 49,000 (36), was demonstrated by immunoblotting in GC. Immunocytochemistry localized the D1-R protein to ovarian cells in vivo and to GC in vitro. Cultured GC posses D1-R-immunoreactivity; both cytoplasmatic and a membrane-associated staining were observed, as well as cells without staining. Thus, not all cells may have functional, membrane-associated receptors. Such a heterogeneity is also suggested by the change of cytomorphology after the addition of DA to GC. DA induced cell contraction in most (but not all) cells, an effect which was not abolished by a ß-receptor antagonist but was duplicated by SKF-38393. Cell contraction was previously described in GC (38), as a result of cAMP elevation. Thus, our results suggest that the observed contraction is cAMP-mediated and indicate that contraction is specifically linked to D1-R rather than ß-adrenergic receptor activation.
Additional results indicate that DA and/or SKF-38393 affect D1-R-bearing targets in cultured GC, resulting in a significant increase in the levels of medium cAMP, the second messenger of D1-R (37). cAMP in GC cultures increased more than 2-fold after addition of 100 µmol/L SKF-38393, a result in the same range as reported for rat ovarian cells (21). This is a small effect, compared with that produced by 10 IU/mL CG, which doubles basal progesterone production and causes a 60- to 80-fold increase in cAMP production (Mayerhofer and Boddien, unpublished). It is therefore conceivable that small changes in cAMP, induced by D1-R activation, may lead to changes in progesterone accumulation in the culture media of GC that were too small to be detected with the methods used. Alternatively, not-yet-defined intracellular cross-talk events, between the signal transduction pathways of hCG/LH-receptors and D1-R, might also account for the lack of stimulation of steroid production in these experiments.
Though production of progesterone is a major function of GC, its regulation seems not to be the target for down-stream events after D1-R activation. We have attempted to identify such a target and have shown expression of DARPP-32, a protein linked to D1-R-bearing cells, in the ovary and in GC (by means of immunocytochemistry, immunoblotting, and RT-PCR-cloning). The sequence obtained is 87% homologous, at the nt level, to the bovine sequence; but the deduced amino acid sequence differs, in two positions, from the sequence previously published for human DARPP-32 (39). These changes were present in all four cDNA clones, but a PCR and/or sequencing artifact cannot be ruled out. DARPP-32 immunoreactive protein was demonstrated in Western blots and in immunocytochemical studies, which showed a cytoplasmic localization of this protein in GC.
Though DARPP-32 is typically present in neurons that possess D1-R, its expression is restricted neither to neurons nor to the central nervous system (30, 38); it has been identified in tanycytes and choroid plexus epithelial cells, as well as in brown fat, parathyroid, and renal epithelial cells (30, 40, 41). After phosphorylation of threonine 34, DARPP-32 can function as a specific and potent inhibitor of protein phosphatase-1 (42). Interestingly, several other first messengers besides DA have been found to influence the phosphorylation state of DARPP-32 [including serotonin, vasoactive intestinal peptide, glutamate, GABA, cholecystokinin, NE (41), and nitric oxide or nitrogen monoxide (43)]. DARPP-32, therefore, represents a multifunctional integrator for several first messengers, at least in the systems examined so far. In the present study, we have only examined the possibility of D1-R-mediated phosphorylation of DARPP-32. We used two different approaches (Western blotting using a phospho-threonine-DARPP-32 antibody; and immunoprecipitation studies using a monoclonal DARPP-32 antibody). DARPP-32 phosphorylation was increased by DA or SKF-38393 within 1 h, using either approach. The increased phosphorylation resulted from D1-R activation, because it was abolished by the D1-R antagonist SCH-23390. The SKF-38393 effect persisted when ß-adrenoreceptors were blocked by propranolol. Phosphorylation of DARPP-32, not only at threonine 34 but at additional sites (presumably including serine 137) may also occur in GC. This was indicated by the migration pattern of phospho-DARPP-32, which resulted in a doublet band in Western blots when probed with the phospho-DARPP-32 monoclonal antibody (32, 34, 35). Interestingly, phosphorylation of DARPP-32 at serine 137 decreases dephosphorylation of threonine 34 and thus may reinforce the inhibitory action of DARPP-32 on protein phosphatase-1 (34). Under control conditions, without adding SKF-38393, phosphorylated DARPP-32 was also detected, indicating that other (as yet unidentified) signals that are present in the medium and/or may be produced by these cells can be active, as well. That DA seems to be among these factors is indicated by the ability of SCH-23390 to reduce the phosphorylation in some cases. The expression of DARPP-32 and its phosphorylation in GC, which also possess receptors for some other first messengers [e.g. serotonin (44), catecholamines (8), and VIP (45)], opens up a new perspective on how DA and other signals may be integrated within this human ovarian cell type.
In neurons and other DARPP-32-expressing cells, only a few of the down-stream targets of protein phosphatase-I have been identified. Among the known targets are proteins responsible for the regulation of the membrane potential, including Na+/K+-ATPase and ion channels (such as Na+-channels and N-, P- and L-type Ca2+-channels) (30, 40). GC possess some potential targets, including Na+/K+-ATPase. They also contain progesterone receptors (46), which, in other systems, can be activated by DA (47, 48). Additional studies are currently being conducted to determine whether those or other proteins are targets for the DA/D1-R/DARPP-32 system in GC and to examine the functional significance of DARPP-32 and the consequences of its phosphorylation in the ovary.
The catecholamines NE and DA can reach the ovary via several routes (see introductory portion of text). DA accumulates in follicular fluid of human preovulatory follicles (16, 17, 18), which contain DA in concentrations as high as 15 µg/100 mL. Because follicles are devoid of blood vessels, nerve fibers, and neuron-like cells, the presence of DA suggests that DA, released from its sources, diffuses through the extracellular space to the follicles. In the follicle, DA may have an indirect and a direct role: thus, as shown most recently, oocytes (though not expressing D1-R) are endowed with a DA transporter, can take up DA, and can synthesize NE (49). NE subsequently can activate ß-adrenergic receptors of follicular cells (49). The present study indicates another, direct (and likewise, physiological) role for DA: DA in vivo is available to those cells that we have identified to possess a D1-R and DARPP-32, i.e. GC. Thus, the localization of the D1-R and DARPP-32 and the presence of DA in the human ovary suggest that this system may participate in the regulation of the complex events associated with follicular development, possibly ovulation, and/or the regulation of the corpus luteum. Additional studies will be necessary to determine the implications of these results for human reproductive medicine. In addition, these results and the growing knowledge about DA-receptors in endocrine adrenal cells (37) and in nonendocrine epithelial cells of the stomach and duodenum (50) indicate that DA may have as-yet-unrecognized functions in organs other than the brain.
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Acknowledgments |
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Footnotes |
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Received July 7, 1998.
Revised September 21, 1998.
Accepted September 23, 1998.
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 K. L. Jones, S. S. King, K. E. Griswold, D. Cazac, and D. L. Cross Domperidone can ameliorate deleterious reproductive effects and reduced weight gain associated with fescue toxicosis in heifers J Anim Sci, October 1, 2003; 81(10): 2568 - 2574. [Abstract] [Full Text] [PDF]
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L. Kunz, A. Thalhammer, F. D. Berg, U. Berg, D. M. Duffy, R. L. Stouffer, G. A. Dissen, S. R. Ojeda, and A. Mayerhofer Ca2+-Activated, Large Conductance K+ Channel in the Ovary: Identification, Characterization, and Functional Involvement in Steroidogenesis J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5566 - 5574. [Abstract] [Full Text] [PDF]
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S. Fritz, L. Kunz, N. Dimitrijevic, R. Grunert, C. Heiss, and A. Mayerhofer Muscarinic Receptors in Human Luteinized Granulosa Cells: Activation Blocks Gap Junctions and Induces the Transcription Factor Early Growth Response Factor-1 J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1362 - 1367. [Abstract] [Full Text] [PDF]
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S. Fritz, I. Wessler, R. Breitling, W. Rossmanith, S. R. Ojeda, G. A. Dissen, A. Amsterdam, and A. Mayerhofer Expression of Muscarinic Receptor Types in the Primate Ovary and Evidence for Nonneuronal Acetylcholine Synthesis J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 349 - 354. [Abstract] [Full Text]
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A. Mayerhofer, S. Fritz, R. Grünert, S. L. Sanders, D. M. Duffy, S. R. Ojeda, and R. L. Stouffer D1-Receptor, DARPP-32, and PP-1 in the Primate Corpus Luteum and Luteinized Granulosa Cells: Evidence for Phosphorylation of DARPP-32 by Dopamine and Human Chorionic Gonadotropin J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4750 - 4757. [Abstract] [Full Text]
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 B. Sommersberg, A. Bulling, U. Salzer, U. Fröhlich, R. E. Garfield, A. Amsterdam, and A. Mayerhofer Gap Junction Communication and Connexin 43 Gene Expression in a Rat Granulosa Cell Line: Regulation by Follicle-Stimulating Hormone Biol Reprod, December 1, 2000; 63(6): 1661 - 1668. [Abstract] [Full Text]
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J. Grosse, A. Bulling, C. Brucker, U. Berg, A. Amsterdam, A. Mayerhofer, and M. Gratzl Synaptosome-Associated Protein of 25 Kilodaltons in Oocytes and Steroid-Producing Cells of Rat and Human Ovary: Molecular Analysis and Regulation by Gonadotropins Biol Reprod, August 1, 2000; 63(2): 643 - 650. [Abstract] [Full Text]
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 A. Bulling, F. D. Berg, U. Berg, D. M. Duffy, R. L. Stouffer, S. R. Ojeda, M. Gratzl, and A. Mayerhofer Identification of an Ovarian Voltage-Activated Na+-Channel Type: Hints to Involvement in Luteolysis Mol. Endocrinol., July 1, 2000; 14(7): 1064 - 1074. [Abstract] [Full Text]
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S. Fritz, K. J. Föhr, S. Boddien, U. Berg, C. Brucker, and A. Mayerhofer Functional and Molecular Characterization of a Muscarinic Receptor Type and Evidence for Expression of Choline-Acetyltransferase and Vesicular Acetylcholine Transporter in Human Granulosa-Luteal Cells J. Clin. Endocrinol. Metab., May 1, 1999; 84(5): 1744 - 1750. [Abstract] [Full Text]
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